Method of screening tumor metastasis suppressor gene using RNA interference library

A technique for RNA interference and tumor metastasis, which is applied in the field of screening tumor metastasis suppressor genes using RNA interference libraries, which can solve the problems of inability to form tumor metastasis, unfavorable screening of tumor metastasis suppressor genes, and elevated

Inactive Publication Date: 2009-06-17
黄行许 +1
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Problems solved by technology

The disadvantage is that after the overexpression of the tumor metastasis suppressor gene, the tumor metastasis caused by the inactivation of the tumor metastasis suppressor gene cannot be formed, so it is not conducive to the screening of the tumor metastasis suppressor gene

Method used

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Examples

Experimental program
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Effect test

specific Embodiment approach

[0056] 1. Using RNA interference library DNA to transfect Phoenix retrovirus packaging cells

[0057] Day 1: Preparing Phoenix Retroviral Packaging Cells

[0058] 1. 18-24 hours before transfection, press 3×10 6 / 10 cm plate inoculation of Phoenix cells onto the plate;

[0059] 2. Let the cells adhere to the wall for 10-24 hours;

[0060] 3. When the cell density reaches 2 / 3, a 10 cm plate has about 5×10 6 cell. At this time packaging cells are most easily transfected and produce the highest titers of virus at 37°C.

[0061] *Medium: DMEM, 10% fetal bovine serum, 1% penicillin-streptomycin, 1% glutamine.

[0062] Day 2: Transfection

[0063] 1. Prepare the DNA for transfection and dissolve it in HEPES solution;

[0064] 2. About 5 minutes before transfection, add 25 μM chloroquine to the plate (the storage concentration of chloroquine is 50 mmol);

[0065] * Chloroquine inhibits DNase in lysosomes by neutralizing the pH inside intracellular transport microvesicles;

...

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Abstract

The invention relates to a method for sieving tumor metastasis inhibiting genes by utilization of an RNA interference library, which is to utilize an in vivo transplantation tumor metastasis model of immunodeficient mouse cells to better simulate generation of tumor metastasis than a cell model, and utilize the RNA interference library to make various genes of a human genome randomly reduce expression in tumor cell strains respectively, so as to greatly strengthen the potency of cells of which the tumor metastasis inhibiting genes reduce expression to form tumor metastasis. Therefore, after cells treated by the RNA interference library are injected into the in vivo of an immunodeficient mouse, the cells of which the tumor metastasis inhibiting genes reduce expression can form a lung tumor in the condition that contrast cells can not form tumor metastasis, and novel tumor metastasis inhibiting genes can be determined and sieved by separating the lung tumor and cloning the expression reduction genes in the lung tumor. The tumor metastasis inhibiting genes are sieved in the whole genome level by the method, and are used: firstly, for further researching generation, development and an action mechanism of tumor metastasis; secondly, as biological markers of tumor for guiding formulation of a tumor treatment solution under the condition of diagnosis, and evaluating the treatment effect and detecting relapse after diagnosis; and thirdly, as a target of tumor targeting treatment to achieve the aim of preventing and treating tumor metastasis is achieved by strengthening the functions of the tumor metastasis genes.

Description

technical field [0001] The invention relates to a method for screening tumor metastasis suppressor genes by utilizing RNA interference library and immunodeficiency mouse cell transplantation tumor metastasis model in vivo. Background technique [0002] Tumor metastasis is the spread of tumor cells from the original site to other parts of the body. Statistics show that more than 90% of cancer patients' deaths are due to tumor metastasis. Tumor metastasis includes: 1. Tumor cells migrate through the basement membrane of tumor tissue; 2. Enter the circulatory system, survive and transport in it; 3. Retain in the secondary tumor site; 4. Leak out from the circulatory system; site and proliferate to form new tumors and other processes. Due to its complexity, despite its extensive study, tumor metastasis remains by far the least understood process of tumor biology. The complex process of tumor metastasis is regulated by a variety of genes. Some of these genes promote the occur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K49/00C12Q1/68
Inventor 黄行许罗如意
Owner 黄行许
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